Novel nucleotide triphosphates as potent P2Y2 agonists

Article abstract of DOI:10.1016/j.bmcl.2006.09.017

The synthesis and P2Y₂ activities of a novel series of nucleoside triphosphates are described. Many of these compounds were potent agonists of the P2Y₂ receptor.

Full text of DOI:10.1016/j.bmcl.2006.09.017

Bioorganic & Medicinal Chemistry Letters 17 (2007) 562–565
Novel nucleotide triphosphates as potent P2Y2 agonists
Daniel Brookings,b Richard J. Davenport,a Jeremy Davis,b Frances C. A. Galvin,a
Steve Lloyd,a Stephen R. Mack,a Ray Owens,ꢀ Verity Sabina,* and Joanne Wynna
aUCB-Group, Granta Park, Abington, Cambridge CB1 6GS, UK
bUCB-Group, 216 Bath Road, Slough SL1 3WE, UK
Received 31 July 2006; revised 6 September 2006; accepted 7 September 2006
Available online 29 September 2006
Abstract—The synthesis and P2Y2 activities of a novel series of nucleoside triphosphates are described. Many of these compounds
were potent agonists of the P2Y2 receptor.
Ó 2006 Elsevier Ltd. All rights reserved.
O
The P2Y receptors are a group of 7-transmembrane G-
O
P
HN
O
P
O
P
O
P
protein coupled receptors that are activated by purine
and pyrimidine nucleotides. Several human subtypes have
been cloned and characterised.1 Activation of P2Y2 recep-
tors in the airway epithelium increases mucus secretions,
cilia beat frequency and transport of chloride ions and
water across the luminal surface, leading to an increase
in the rate of mucociliary clearance. In the conjunctiva
P2Y2 activation stimulates tear secretion from lacrimal
tissues. A P2Y2 agonist may therefore provide a useful
therapy in diseases such as cystic fibrosis and dry eye.2
O
O
O
O
N
O
O
R1
O
O
OH OH
HO HO
R
HO
HO OH
NH2
N
O
INS37217
R = OH, R1 =
INS365
R = OH, R1 =
NH
N
O
N
O
O
Our objective was to identify some novel P2Y2 agonists
with potencies similar to that of UTP. We therefore pre-
pared some UTP analogues in which the triphosphate
and ribose units were retained, but incorporating some
‘unnatural’ bicyclic aromatic bases in place of the uracil.
O
P
NH
O
P
HO
O
P
UTP
O
HO
O
O
O
N
O
OH OH
HO OH
A group of available 6,6 and 6,5 fused bicyclic pyridones
was reacted with bis(trimethylsilyl)acetamide in acetoni-
trile, followed by tribenzoyl protected ribofuranose ace-
tate and tin tetrachloride. The products of this reaction
were treated with sodium methoxide in methanol to re-
move the benzoyl esters, and the resulting nucleosides
converted to triphosphates using standard conditions.5
The desired triphosphates were separated from the crude
reaction mixtures by preparative HPLC and isolated as
the ammonium salts (Scheme 1). Incorporation of the
triphosphate unit was confirmed by LC-MS and 31P
NMR spectroscopy. The 31P NMR pattern characteris-
UTP is a potent natural agonist for the P2Y2 receptor,
but is chemically unstable and readily metabolised and
therefore has a very short duration of action. Various
dinucleotide polyphosphates have been investigated
as purinergic agonists with improved stability over
UTP.3 The P2Y2 agonists INS365 (diquafosol tetrasodi-
um) and INS37217 (denufosol tetrasodium) are in
development for dry eye disease and cystic fibrosis,
respectively.4
Keywords: P2Y2; SAR; Potent agonists; Stability.
*
tic of
a triphosphate (doublet, doublet, triplet,
Corresponding
author.
verity.sabin@UCB-group.com
Fax:
+44
1223
896400; e-mail:
J = 20 Hz) could clearly be seen in each case (Fig. 1).
The P2Y2 agonist potencies of the resulting triphos-
phates are shown in Table 1.
ꢀ
Present address: Wellcome Trust Centre for Human Genetics,
University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.017

D. Brookings et al. / Bioorg. Med. Chem. Lett. 17 (2007) 562–565
563
BzO
The isocarbostyril 4a, pyridyl pyridone 4b and thienyl
pyridone 4e were all sub 50 nM agonists of P2Y2. The
partially reduced compound 4c and the furanyl pyridone
4d were only slightly less potent, while 4f, the regioisom-
er of 4d, was significantly less active.
R
O
i, ii
RH
2a-f
1a-f
BzO
OBz
iii
We next investigated the positional effect of a methyl
group around the isocarbostyril template. 4-Methyl iso-
carbostyril was prepared by the reaction of allyl amine
with 2-iodobenzoyl chloride followed by a palladium-
catalysed cyclisation, while 5-, 6-, 7- and 8-methyl iso-
carbostyrils were prepared7 from cinnamic acids via a
Curtius rearrangement or by rearrangement of isoquin-
oline-N-oxides (Scheme 2). These were converted to the
nucleotide triphosphates 5a–5e following the procedure
in Scheme 1.
PPPO
HO
iv, v, vi
R
O
R
O
4a-f
3a-f
HO
OH
HO
OH
Scheme 1. Reagents: (i) BSA, MeCN, (ii) b-O-ribofuranose-1-acetate-
2,3,5-tribenzoate, SnCl4; (iii) NaOMe, MeOH; (iv) POCl3, P(OMe)3,
proton sponge; (v) H4P2O7Æ1.5(Bu3N), Bu3N, DMF; (vi) NH4Cl, H2O.
The P2Y2 agonist potencies of these compounds are
shown in Table 2. Methylation at the 4-position was
very detrimental to activity. The 6-, 7- and 8-positions
were tolerant of substitution, though in no case was
potency improved compared with the unsubstituted
compound 4a.
To explore the SAR around the 6- and 7-positions, some
isocarbostyrils with a variety of 6- and 7- substitutents
were prepared7 from cinnamic acids or isoquinoline-N-
oxides, and converted to the nucleotide triphosphates
5f–5p as described in Scheme 1. The P2Y2 agonist poten-
cies of these compounds are shown in Table 3. The only
examples not to be prepared directly from the appropri-
ate substituted isocarbostyrils were the cyano com-
pounds, prepared by treating the appropriate
tribenzoyl-protected bromo-substituted isocarbostryil
nucleosides with copper cyanide in DMF, and the sul-
fone, by oxidation of the thioether. Some disubstituted
compounds were prepared from the 6,7-difluoro isocar-
bostyril nucleoside by nucleophilic displacement of the
6-fluorine (Scheme 3).
Figure 1. 31P NMR spectrum of a triphosphate.
Although the 6-methyl compound 5c was significantly
more active than the 7-methyl compound 5d, it was
found that substitution with fluoro, chloro and cyano
in the 6-position (5f, 5g, and 5h) gave less active com-
pounds than the corresponding 7-isomers (5i, 5j, and
5k). These offered excellent agonist potencies, compara-
ble to that of UTP. The 7-methoxy compound 5l gave
no advantage over the unsubstituted isocarbostyril 4a,
and substituting at the 7-position with a thioether or a
sulfone (5m and 5n) reduced potency. However, 6-,7-di-
substitution with a thioether, dimethyl amino or meth-
oxy group at the 6-position and fluorine at the
7-position (5q, 5r, and 5s) again provided compounds
with potencies similar to that of UTP; these were some
of the most active P2Y2 agonists synthesised in our
laboratory.
Table 1. Agonist potencies of compounds 4a–4f compared with UTP
and INS365
N
N
N
N
N
O
c
a
b
O
O
O
O
S
N
N
O
d
f
e
O
O
6
Compound
P2Y2 EC50 (nM)
4a
4b
31
35
55
7
4c
UTP
Some key compounds were tested against three other
purinergic receptor subtypes, P2Y1, P2Y4 and P2Y6.
The results of these experiments are shown in Table 4.
4d
4e
68
30
4f
INS365
221
1004a
Whilst UTP has agonist activity at P2Y4 and P2Y6 recep-
tors, and INS365 at P2Y4, our isocarbostyril-substituted

564
D. Brookings et al. / Bioorg. Med. Chem. Lett. 17 (2007) 562–565
I
I
ii
i
HO
HN
HN
O
O
O
HO2C
iii, iv
R
R
v
HN
O
vi
vi, vii
viii
R
R
R
N
N+
HN
-
O
O
Scheme 2. Reagents and conditions: (i) allyl amine, Et3N, DCM; (ii) Pd(PPh3)4, Et3N, Bu4NCl, DMF; (iii) Et3N, ethyl chloroformate, acetone, 0 °C;
(iv) NaN3, H2O; (v) (C6H5)2CH2, Bu3N, toluene, 190 °C; (vi) m-CPBA, DCM; (vii) Ac2O, reflux; (viii) NaOH, H2O.
Table 2. Agonist potencies of compounds 5a–5e
BzO
R
O
4
5
N
6
R
3
PPPO
O
i
N
O
R = CN
R = Br
7
BzO
OBz
8
ii
O
5a-5e
R = SMe
R
R = SO2Me
HO
OH
6
Compound
R
P2Y2 EC50 (nM)
HO
O
N
5a
5b
5c
5d
5e
4-Me
5-Me
6-Me
7-Me
8-Me
17,500
557
35
F
O
iii
iv
v
HO
OH
R = SMe
R = NMe2
R = OMe
R = F
R = F
R = F
148
85
Scheme 3. Reagents and condition: (i) CuCN, DMF, reflux; (ii) m-
CPBA, DCM; (iii) NaSMe, EtOH, reflux; (iv) Me2NH, H2O, 90 °C; (v)
NaOMe, MeOH, reflux.
Table 3. Agonist potencies of 6- and 7-substituted isocarbostyrils 5f–5s
Table 4. Agonist potencies against some other P2Y receptor subtypes
R1
8
8
8
Compound
P2Y1 EC50
(nM)
P2Y4 EC50
(nM)
P2Y6 EC50
(nM)
PPPO
N
O
R2
UTP
INS365
4a
>2000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
39
4004a
7043
424
>20,0004a
>20,000
>20,000
>20,000
>20,000
>20,000
O
5f-5s
HO
OH
5i
3583
Compound
R1
R2
P2Y2 EC50 (nM)
6
5j
5p
>20,000
>20,000
>20,000
5f
F
Cl
H
H
H
F
77
541
88
5
5q
5g
5h
5i
CN
H
nucleotide triphosphates had little or no activity against
these isoforms and thus provide a more selective template
than the uracil-derived P2Y2 agonists. All compounds
tested were inactive against P2Y1.
5j
H
Cl
10
12
26
140
650
13
16
3
5k
5l
H
CN
OMe
SMe
SO2Me
Cl
H
5m
5n
5o
5p
5q
5r
5s
H
H
Cl
In conclusion, a selection of novel UTP analogues bear-
ing ‘unnatural’ bicyclic bases in place of the uracil have
been synthesised and their P2Y2 agonist potencies inves-
tigated. Several of these compounds had activity compa-
rable to that of UTP, and some key examples showed
F
F
SMe
NMe2
OMe
F
F
4
F
6

D. Brookings et al. / Bioorg. Med. Chem. Lett. 17 (2007) 562–565
565
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excellent selectivity for P2Y2 over P2Y4 and P2Y6. Our
isocarbostyril nucleotide triphosphate template thus
provided some novel, potent and selective P2Y2
agonists.
4. (a) Pendergast, W.; Yerxa, B. R.; Douglass, J. G.; Shaver,
S. R.; Dougherty, R. W.; Redick, C. C.; Sims, I. F.;
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Acknowledgments
The authors thank Slough and Cambridge NCE Project
Biology groups and Mr. P. J. Gilbert for their contribu-
tions to this work.
6. Human P2Y2 receptor was isolated from placental cDNA
by PCR and expressed in a human astrocytoma cell line,
1321N1. The calcium response assay was performed in a
FLIPRTM. Values are means of at least three experiments.
7. Briet, N.; Brookes, M. H.; Davenport, R. J.; Galvin, F. C.
A.; Gilbert, P. J.; Mack, S. R.; Sabin, V. Tetrahedron 2002,
58, 5761.
8. Cloned Human P2Y1, P2Y4, and P2Y6 receptors were
stably expressed in 1321N1 cells and assayed as described
for P2Y2. Values are the means of at least four experiments.
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G.; Brown, E. G.; Boyer, J. L.; Patel, R. I.; Redick, C. C.;